Endotoxin binding and neutralizing protein and uses thereof

ABSTRACT

Endotoxin binding/neutralizing proteins capable of binding endotoxin in vivo, thereby neutralizing the toxic effect or bioactivity of endotoxin which are isolated from a horseshoe crab such as  Limulus polyphemus,  pharmaceutical compositions and pharmaceutical uses of the proteins, a method of purifying the proteins and an assay for endotoxin based on the proteins, are disclosed.

[0001] This application is a continuation-in-part of application Ser.No. 07/701,501, filed May 16, 1991, which is a continuation-in-part ofapplication Ser. No. 07/480,957, filed Feb. 16, 1990, which is adivisional of application Ser. No. 07/210,575, filed Jun. 23, 1988.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention is directed to pharmaceutical compositionsand uses of an endotoxin binding/neutralizing protein which may beisolated from a horseshoe crab. The invention covers, inter alia,pharmaceutical compositions and pharmaceutical uses of the protein, amethod of purifying the protein, and an assay for endotoxin based onthis protein.

[0004] 2. Discussion of the Background

[0005] Despite aggressive management, septic shock arising fromgram-negative sepsis continues to be a leading cause of death in bothsurgical and medical patients. Death in such patients usually resultsfrom cardiovascular collapse and/or multiple organ system failure. Oneof the main components of gram-negative bacteria thought to play anintegral role in causing septic shock is an outer wall constituent,endotoxin.

[0006] Endotoxins are high molecular weight complexes, associated withthe outer membrane of gram-negative bacteria, that produce pyrogenicreactions upon intravenous administration. Endotoxin is shed from livingbacteria and is also released into the environment when bacteria die anddecompose. Since gram-negative bacteria are found in great numbers inair, water, and soil, bacterial endotoxin commonly contaminates rawmaterials and processing equipment used in the manufacturing of, forexample, pharmaceuticals.

[0007] Bacterial endotoxin is a complex consisting of lipid,carbohydrate and protein. It is characterized by an overall negativecharge, heat stability and high molecular weight. Highly purifiedendotoxin does not contain protein, and is a lipopolysaccharide (LPS).Depyrogenation can generally be achieved by inactivating or removingendotoxin, depending upon the physicochemical nature of the LPS. LPSconsists of three distinct chemical regions, lipid A, which is theinnermost region, an intermediate core polysaccharide, and an outermost0-specific polysaccharide side chain which is responsible for anendotoxin's particular immunospecificity.

[0008] Bacterial endotoxins are known to have profound biologicaleffects in animals and humans, and to cause severe medical problems whenpresent. Symptoms include induction of high fever, activation ofcomplement, and hypotension. It is critical to avoid endotoxincontamination in any pharmaceutical product or medical device whichcomes into contact with body fluids. High endotoxin levels in sera dueto bacterial diseases, such as septicemia, are not easily treated.Antibiotic treatment of the infection only kills the bacteria, leavingthe endotoxin from their cell walls free to cause fever.

[0009] The horseshoe crab Limulus polyphemus is particularly sensitiveto endotoxin. The cells from their hemolymph (amebocytes) undergo acomplex series of biochemical reactions resulting in clot formation,analogous to mammalian blood coagulation. This phenomenon has beenexploited in the form of bioassays sensitive to very low endotoxinlevels. Currently, a bioassay of this type is the method of choice formonitoring pharmaceutical manufacturing and is termed Limulus AmebocyteLysate (LAL). See U.S. Pat. Nos. 4,276,050, 4,273,557, 4,221,866,4,201,865, 4,038,147, 3,944,391 and 3,915,805, each of which isincorporated herein by reference.

[0010] It has long been observed that once endotoxin interacts with LALthe toxin is not recoverable from the clot. See Nachum et al, Journal ofInvertebrate Pathology, 32:51-58 (1978). This observation ledinvestigators to postulate two alternative explanations. Either theendotoxin is enzymatically degraded during clot formation or it is boundby some factor causing it to lose toxicity. The present inventorsinitiated experiments to extract the endotoxin inactivating factor fromthe LAL.

[0011] Other research groups have experimented with endotoxin bindingproteins, also referred to as anti-LPS factor. To the inventor'sknowledge, the following publications resulting from work in this areaare the most relevant to this invention:

[0012] Tanaka et al, Biochem. Biophys. Res. Comm. 105, 717-723 (1982),

[0013] Iwanaga et al, International symposium on Pyrogen, 84-84 (Jun.23-26, 1987),

[0014] Aketagawa et al, J. Biol. Chem. 261, 7354-7365 (1986),

[0015] Hao, U.S. Pat. No. 4,677,194 (Jun. 30, 1987), and

[0016] Nachum et al, J. Inv. Path. 32, 51-58 (11978).

[0017] Tanaka et al, Iwanaga et al, and Aketagawa et al each conductedresearch on an anti-LPS factor or endotoxin binding protein isolatedfrom a horseshoe crab system. Based on experimental work done in theinventors' laboratory, it appears that a protein involved in the presentinvention is the same as that isolated by Iwanaga et al and Tanaka etal. However, these publications do not say anything about pharmaceuticalutility of the endotoxin binding/neutralizing protein, and it isdifficult to predict in vivo activity based on in vitro experimentation.In fact, neither of these references suggests a practical utility forthe anti-LPS factor, and in view of the unpredictable nature of in vivoactivity, it has previously not been appreciated that the endotoxinbinding/neutralizing protein could be used in a pharmaceuticalcomposition. Furthermore, none of the references disclose the use of theendotoxin binding/neutralizing protein for an endotoxin assay, asdisclosed in the present invention. It is notable that the presentinventors have also discovered certain endotoxin binding/neutralizingprotein variants which have amino acid structures that are differentfrom the anti-LPS factor disclosed in the above-described publications.

SUMMARY OF THE INVENTION

[0018] Accordingly, it is an object of the present invention to providepharmaceutical compositions containing therein anendotoxin-binding/neutralizing protein isolated from a horseshoe crab,which compositions are capable of binding and neutralizing endotoxin invivo.

[0019] It is yet another object of this invention to providepharmaceutical compositions capable of binding and neutralizingendotoxin in vivo and containing therein an endotoxinbinding/neutralizing protein corresponding at least to part of theendotoxin binding and neutralizing domain of the endotoxinbinding/neutralizing protein isolated from a horseshoe crab inaccordance with the invention.

[0020] It is yet another object of the present invention to provide amethod for reducing endotoxin concentration and/or neutralizingendotoxin activity in vivo.

[0021] It is yet another object of the present invention to provide amethod for purifying an endotoxin binding/neutralizing protein from ahorseshoe crab.

[0022] It is yet another object of the present invention to provide amethod of assaying for endotoxin in a fluid.

[0023] These and other objects of the present invention which willhereinafter become more readily apparent, have been provided bypurifying an endotoxin binding/neutralizing protein from the horseshoecrab Limulus Polyphemus, and discovering that it has the capability ofbinding to and neutralizing endotoxin in vivo. It has also beenrecognized that this purified endotoxin binding/neutralizing protein isuseful in an assay for endotoxin. The present inventors have alsodiscovered that there are certain structural variants of the Limuluspolyphemus endotoxin binding/neutralizing protein, and it is expectedthat these materials will also possess endotoxin binding/neutralizingability such that they may be used in the other aspects of the presentinvention as well.

[0024] The present inventors have also discovered a domain within theendotoxin binding protein which is necessary for endotoxinbinding/neutralizing. The present invention thus uses a proteincorresponding at least to this endotoxin binding/neutralizing domain andup to the complete protein sequence.

BRIEF DESCRIPTION OF THE FIGURES

[0025] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

[0026]FIG. 1 shows cation exchange chromatographic purification ofLimulus endotoxin binding protein using CM-Sepharose resin. Peak 1: flowthrough, Peak 2: 0.25M NaCl elution, Peak 3: 1 M NaCl elution.

[0027]FIG. 2 shows reversed phase chromatographic purification of Peak−1) from FIG. 1. Peak A: 25% isopropanol (IPA) elution, Peak 3: 0.35%IPA elution, Peak C: 50% IPA elution, Peak D: column wash with 100% IPA.

[0028]FIG. 3 is a plot of apparent endotoxin concentration (measured byLAL) versus protein concentration. Such a plot can be used to assessprotein specific endotoxin inactivating/neutralizing activity. Specificactivity is expressed as the amount of protein needed to achieve 50%reduction of endotoxin activity in the assay system (5 nanograms ofendotoxin Per 100 microliters). For convenience, units are expressed asmicrograms protein×10⁵ needed to reduce 5 nanograms activity/100microliters of standard endotoxin by 50%.

[0029]FIG. 4 shows endotoxin removal by affinity chromatography withendotoxin binding/neutralizing protein immobilized to a solid support.

[0030]FIG. 5 shows fluorescence excitation and emission spectra ofLimulus endotoxin binding/neutralizing protein.

[0031]FIG. 6 shows successive decreases in the emission spectra ofLimulus endotoxin binding/neutralizing protein upon successive additionsof endotoxin.

[0032]FIG. 7 shows lack of successive decreases in the emission spectraof human serum albumin upon successive additions of endotoxin (negativecontrol).

[0033]FIG. 8 shows lack of successive decreases in the emission spectraof peak B, FIG. 2 upon successive additions of endotoxin (negativecontrol).

[0034]FIG. 9 shows a fluorescence titration of Limulus endotoxinbinding/neutralizing protein at pH 3.86. K_(D)=2.41 micromolar.

[0035]FIG. 10 shows fluorescence titration of Limulus endotoxinbinding/neutralizing protein at pH 6.91. K_(D)=0.51 micromolar.

[0036]FIG. 11 shows fluorescence titration of Limulus endotoxinbinding/neutralizing protein at pH 8.80. K_(D)=2.40 micromolar.

[0037]FIG. 12 shows the effect of pH on dissociation constant of Limulusendotoxin binding/neutralizing protein.

[0038]FIG. 13 is a comparison of immobilized Limulus endotoxinbinding/neutralizing protein compared to two immobilized polymyxinresins in proteinaceous and non-proteinaceous solutions.

[0039]FIG. 14 demonstration of activity of Limulus endotoxinbinding/neutralizing protein in the presence of total human serum. Thedashed line represents serum alone as a control (68 nanograms ofendotoxin per ml inactivated/neutralized). The solid line representsserum plus 25 micrograms/ml added endotoxin binding/neutralizing protein(1600 nanograms endotoxin per ml inactivated/neutralized).

[0040]FIG. 15 provides the amino acid sequence of the endotoxinbinding/neutralizing protein isolated from Limulus polyphemus inaccordance with the present invention (SEQ. ID. NO. I).

[0041]FIG. 16 sets forth a DNA sequence (SEQ. ID. NO. II) encoding aprotein corresponding to the endotoxin binding/neutralizing protein ofthe present invention having attached to its amino terminus thetetrapeptide Glu-Ala-Glu-Ala. The so modified endotoxinbinding/neutralizing protein is SEQ. ID. NO. III. This DNA sequence isequipped with yeast preferred codons and possesses unique restrictionenzyme recognition sites for convenient modification of the sequence.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The endotoxin binding/neutralizing protein of the presentinvention may be isolated from any horseshoe crab. For example, any ofthe four known species of horseshoe crabs could be used. These speciesare:

[0043]Limulus polyphemus

[0044]Tachypleus gigas

[0045]Tachypleus tridentatus

[0046]Carcinoscorpius rotundicauda

[0047] Especially preferred among these is Limulus polyphemus, thehorseshoe crab which is found along the North American coast. Theendotoxin binding/neutralizing protein may be isolated by a procedurewhich is summarized hereinbelow. The procedure is illustrated for theLimulus horseshoe crab, but these procedures may be applied to any knownhorseshoe crab, as recited herein.

[0048] It was surprisingly found that the cellular debris producedduring lysate production from Limulus amebocytes contains significantamounts of the endotoxin binding/neutralizing protein. The cellulardebris was found to have even more activity than lysate itself. By “celldebris” is meant the insoluble material remaining when Limulusamebocytes are lysed by hypotonic shock. It includes nuclei, cell debrisand some lysate proteins. The majority is insoluble material. Hypotonicshock may be accomplished by treating the Limulus amebocytes withendotoxin-free distilled water, preferably at 4° C., with shaking (e.g.,for 12 h). The mixture is centrifuged, separating the lysate (solublesupernatant) from the cell debris.

[0049] The next step is to extract from the cell debris the Limulusamebocyte binding protein. Many solvents have been tested, includingwater, several alcohols (e.g., methanol, ethanol, isopropanol, andbutanol), acetone, chloroform, acetonitrile, acids (e.g., HCl andH₂SO₄), bases (e.g., NaOH and ethanolamine), salts (e.g., NaCl), anddetergents (e.g., Tween, triton X-100, and SDS). The best results wereobtained with denaturants such as urea and guanidine hydrochloride.Surprisingly, six molar solutions of denaturants were effective inextracting the protein without affecting biological activity. Thisconcentration of denaturant would be expected to inactivate mostproteins. Thus, the first step in the purification procedure herein isextraction of cellular debris from Limulus amebocyte lysate with adenaturant to produce an extract. The concentration of the denaturantcan range from 1 M to 10 M. A more preferred range would be 3 M to 8 M.A most preferred concentration is around 6 molar. Urea is the preferreddenaturant, because it can be made free of endotoxin by ultrafiltrationand it is not readily contaminated by endotoxin containing bacteria.

[0050] Preferably, prior to loading the extract onto a cation exchangecolumn, an ultrafiltration step is performed. This step is alsoaccomplished using urea or another denaturant as described above forextraction. In particular, the extract from the cell debris is crudelyfiltered with a filter aid such as diatomaceous earth (e.g., Celite,Manville Corp.) or one of the cationic or anionic polymeric particles incolloidal suspension (e.g., Biocryl, Supelco division of Rohm and HaasCorp.), then passed through an ultrafiltration membrane. Ultrafiltrationmembranes may be used in any known form (e.g., plain film, hollow fiber,tubular and spiral) and may be made of any material. Preferred materialsare polysulfone, polyvinylidene fluoride, polyacrylonitrile, nylon, orcellulose. Most preferred is a polysulfone, flat or hollow fiber typeultrafiltration membrane. The preferred molecular weight cut-off of theultrafiltration membrane is 20,000 daltons to 50,000 daltons as can becommercially obtained from Millipore, Filtron or other membrane filtermanufacturers. The most preferred membrane has a 30,000 dalton cut-off.The Limulus endotoxin binding/neutralizing protein is now in thefiltrate at a very low concentration. The filtrate from the firstmembrane is concentrated using a second ultrafiltration membrane havinga molecular weight cut-off of 5,000 to 10,000 daltons, preferably an8,000 dalton cut-off membrane. The second ultrafiltration membrane maybe the same or different material as the first ultrafiltration membrane,and is preferably made of polysulfone. The principle of operation is thesame as the 30,000 cut-off membrane, however, the endotoxinbinding/neutralizing protein is now in the retentate with all otherproteins greater than e.g., 8,000 daltons.

[0051] After the above step, the retentate is subjected to cationexchange chromatography using cellulose, cross-linked agarose orhydrophilic-vinyl polymer resins derivatized with carboxymethyl (CM),Sulphopropyl (SP), Sulphonate (S), or other anionic group. These can beobtained from several manufacturers including Pharmacia (Sephadex®,Sepharose®), Tosoh Corp. (Toyopearl®) or BioRad (Cellex®, Bio-Gel). Themost preferred resin is CM-Sepharose®. The pH of loading and elutionbuffers can range from 4 to 8, with the most preferred range of pH 5 to5.5. In this step also, urea (or another denaturant as described abovefor extraction) is an important constituent. All column elution buffersmust contain urea (at least about 3 molar) to elute the endotoxinbinding protein cleanly from the column.

[0052] The elution from the column is accomplished by a step gradient ofa salt such as ammonium chloride, potassium chloride or sodium chloride.Sodium chloride is preferred. FIG. 1 shows the results of the CMSepharose® chromatographic step. The preferred concentration of urea inthe eluent used in this step is 1 M to 6 M. A preferred concentration ofurea in this step is 2 M to 4 M. A most preferred concentration is 2.5 Mto 3.5 M. When sodium chloride is used in the eluant, biologicalactivity elutes at a concentration of sodium chloride of from around 0.5to 1 molar.

[0053] After the cation exchange step, the salt-eluted peak of activityis applied to a reversed-phase column. The preferred method employs aresin having 4, 8, or 18 carbon chains (C-4, C-8 or C-18, respectively).The most preferred method employs a C-4 resin commercially availablefrom such resin manufacturers as Vydac, Waters, Tosoh Corp., etc. Theprotein is eluted by a step gradient of, for example, isopropanol andtrifluoroacetic acid (TFA). The concentration of trifluoroacetic acid isideally 0.2%, but can range from 0 to 0.4%, preferably 0.15 to 0.25%.FIG. 2 shows the location of the activity during treatment on thereversed-phase column. The reversed-phase column step effects desaltingand further purifies the material to virtual homogeneity. The Limulusprotein is remarkably stable to the organic solvents and low pH of thiscolumn. The protein is stable over a pH range of 1-3 in the presence ofTFA. The material may now be lyophilized. The purified Limulus proteinhas an isoelectric pH (pI), which is greater than 10, indicating a verybasic protein. The molecular weight of the protein as determined bySDS-PAGE is 12,500±100. The first three N-terminal amino acids of thispurified material are

[0054] Asp-Gly-Ile

[0055] This protein is the preferred protein used in accordance with thepresent invention. Its amino acid sequence is set forth in FIG. 15 whereit is identified as SEQ. ID. NO. I.

[0056] The inventors have also discovered a domain, within thisendotoxin binding/neutralizing protein, necessary for endotoxinbinding/neutralization. This domain comprises the amino acid sequence offrom amino acid position 30 to amino acid position 55 set forth in FIG.15. Thus in a preferred embodiment, the present invention uses a proteincorresponding at least to that portion of SEQ. ID. NO. I or IIIcorresponding to amino positions 30 to 55 and up to complete SEQ. ID.No. I or III where the components attached to the amino end or thecarboxyl end of the domain may be independently present at varyinglengths.

[0057] It should be noted that Watson and Sullivan, U.S. Pat. No.4,107,077, teaches that there is an increase in sensitivity of thelysate by organic extraction with chloroform. It appeared logical that aprotein which inactivated endotoxin would appear as an inhibitor in theendotoxin assay. Thus, it was hoped that the protein was the inhibitorwhich is extracted into chloroform and could be recovered from thatextract. However, surprisingly, this was not successful. Since theprotein is fairly hydrophobic, it may be in the organic extract, butdenatured in some way.

[0058] The majority of the purified material had an amino acid sequencewhich appears to be identical to a protein from Limulus that wasisolated by Tanaka et al. The material was subsequently purified byIwanaga et al, as reported in the International Symposium on Pyrogen,held Jun. 23-26, 1987 in China. However, in the latter case, a differentpurification procedure was employed.

[0059] In addition to a protein having an apparently identical aminoacid sequence to that reported in Iwanaga et al, some related proteinswere also purified, which have different amino acid sequences. Thesevariant proteins have not previously been described. In a first protein,on the N-terminal thereof, a serine rather than an aspartic acid residueis located. Furthermore, an asparagine is located in the second positionfrom the N-terminal rather than a glycine as reported in Iwanaga et al.Therefore, a protein having an initial amino acid sequence of

[0060] Ser-Asn-Ile-Trp-Thr

[0061] is also part of the present invention. Other possible proteinderivatives are those beginning with

[0062] Asp-Asn-

[0063] Ser-Gly-, and

[0064] Ser-Asn-,

[0065] and also a protein having an N-terminal asparagine with one lessamino acid than the natural sequence. Each of these variant proteins isalso part of the present invention, and they may be used in each aspectof the invention described hereinbelow. They will be referred to asendotoxin binding/neutralizing protein variants (or variant proteins forshort), as distinguished from the natural sequence endotoxin bindingprotein, which is the protein having the amino acid sequence reported inIwanaga et al.

[0066] The variant proteins were not separated from each other. Rather,upon sequencing the most purified samples, some positions were uniformlyone amino acid, while other positions showed major variation. This isinterpreted as a mixture of proteins showing microheterogeneity. It isconsistent with the existence of a gene family for these proteinsindividual gene sequences are very homologous, but not identical. Itremains to be seen if their individual specific activities varysignificantly.

[0067] The present invention also encompasses DNA sequences which encodethe polypeptides of the present invention, particularly those encodingat least the domain of from amino acid position 30 to 55 of SEQ. ID. NO.I or III and up to the whole SEQ. ID. NO. I or III, vectors containingthese DNA sequences, and microorganisms such as E. coli, yeast, etc.,transformed with the vectors. The transformed microorganisms can be usedto produce large quantities of the polypeptide materials, including thevariant proteins described above. The genes of this invention may bealtered so as to maximize codon expression in a given host.

[0068] The DNA sequences used in accordance with the present inventionmay be obtained in any manner known in the art, such as cloning and/orDNA synthesis. Various methods for synthesizing both DNA and RNAsequences are discussed in “Synthesis and Applications of DNA and RNA”,edited by S. A. Narang, Academic Press Inc. (1987), which is herebyincorporated by reference. Once obtained these gene sequences may beexpressed in microorganisms using known methodology. See, e.g., Maniatiset al, “Molecular Cloning: A Laboratory Manual” Cold Spring HarborLaboratory (1982), which discusses cloning and expression methodologiesand which is hereby incorporated by reference.

[0069] In accordance with the preferred embodiment of the presentinvention, a protein encoded by SEQ. ID. NO. III is produced in a yeasthost where it may be produced as a glycoprotein comprising the aminoacid sequence of the endotoxin binding protein of the present invention(SEQ. ID. NO. I) to which the tetrapeptide “Glu-Ala-Glu-Ala” iscovalently attached to the amino terminal of the endotoxin bindingprotein. This glycoprotein encoded by SEQ. ID. NO. II is excreted by theyeast host.

[0070] Procedure for Assaying for Endotoxin Binding Activity

[0071] To follow the purification of the endotoxin binding/neutralizingprotein during the above purification steps, assays can be performed insolution. One publication describing typical assays for LR₅₀ values isNovitsky et al, J. Clin. Microbiol. pp. 211-216 (1985). A specificprocedure follows:

[0072] A 96-well microtiter plate is used. Protein fractions areserially diluted across the top row and mixed with a standard endotoxinsolution. There is another series of dilutions done on all samples toget within range of the assay, but basically the endotoxin recovered inthe well after mixing with a suspected endotoxin inactivating protein ismeasured. This is compared to negative controls and an endotoxinstandard curve. It is found that the added endotoxin is inactivated atthe high protein concentrations. As the protein is diluted across theplate, a point is reached where it is too dilute to inactivate the addedendotoxin. This dilution is a measure of the protein specific activity.FIG. 3 represents a graphical form of such experiments. In FIG. 3, thedotted curve represents the crude extract after ultrafiltration, and thesolid line is the most highly purified fraction from the reversed-phasecolumn.

[0073] In Vivo Activity of the Endotoxin Binding/Neutralizing Protein ofthe Present Invention

[0074] The endotoxin binding protein of the present invention (andvariants thereof) may be incubated with endotoxin, and then administeredto animals, and the bound endotoxin is not found to cause anypyrogenicity in vivo. The details of the endotoxin test are presentedhereinbelow in the examples section.

[0075] Surprisingly, and even less predictably, the present endotoxinbinding protein may be administered to an animal after the animal hasalready been exposed to endotoxin, and the endotoxin binding protein canreverse the effects of free endotoxin in vivo. Moreover, the endotoxinbinding/neutralizing protein may be administered to an animal beforecontact with endotoxin by the animal, and the endotoxinbinding/neutralizing protein will exert a protective effect against theeffects of endotoxin. Accordingly, the endotoxin binding/neutralizingprotein of the present invention may be formulated into a pharmaceuticalcomposition for treating an animal in vivo, so as to exert a therapeuticeffect if endotoxin is present in the animal, or to exert a protectiveor preventive effect, if the animal should come into contact withendotoxin later. Details on the experimental tests showing the in vivoeffects of the endotoxin binding/neutralizing protein of the presentinvention are also presented in the examples section hereinbelow.

[0076] It should be noted that in vivo activity of this type would havebeen unpredictable based on the bare disclosure of the in vitroendotoxin binding/neutralizing capability of this protein. According toNachum (described above), there were two possibilities for the removalof endotoxin by lysate proteins, binding or enzymatic cleavage ofendotoxin. The inventors looked for degradation products and found none(specifically, free fatty acids that could have been released byesterases). Subsequent work has found binding to be the mechanism. It ismore expected that the endotoxin would be inactivated in vivo by anenzymatic action the molecule would be structurally different. However,by only binding, one would assume the entire endotoxin structure wouldstill be present and available to trigger the normal biological responseto the toxin. The noted inactivation in vitro might easily have been anartifact of an artificial assay system—perhaps a conformational changein the bound endotoxin not reacting with the LAL reagent, or it is alsopossible that the Limulus endotoxin binding protein was inhibiting theclotting reaction by itself (since it is present in lysate, thispossibility is hard to rule out without the in vivo results).

[0077] Although it might be possible for the endotoxin binding proteinof the present invention to be administered in vivo without anyadditives thereto, it is preferably mixed with a carrier, and ifnecessary, other adjuvants.

[0078] By the term “carrier” as used herein is meant a synthetic ornatural, inorganic or organic substance which is added to the endotoxinbinding protein of the present invention to assist the active ingredientin reaching the location to be treated therewith and to facilitatestorage, transportation and handling of the active ingredient.

[0079] Among suitable liquid carriers, there may be included aromatichydrocarbons such as benzene, toluene, xylene, cumene, etc., paraffinichydrocarbons such as mineral oil and the like, halogenated hydrocarbonssuch as carbon tetrachloride, chloroform, dichloroethane and the like,ketones such as acetone, methyl ethyl ketone, etc., ethers such asdioxane, tetrahydrofuran and the like, alcohols such as methanol,propanol, ethylene glycol and the like, dimethyl formamide,dimethylsulfoxide, water, etc. Mixtures of any number of liquid carriersare also envisioned. Upon dissolution of lyophilized active ingredientwith unbuffered pyrogen free distilled water or saline or phosphatebuffered saline (pH 6.5 to 7.5), the protein is not completely soluble.In order to avoid undesirable physiological side-effects which mightresult from such suspensions, the pH may be adjusted to slightlyalkaline pH (pH 8 to 9) at which the material becomes water clear. Forthis reason, the most preferred liquid carrier is pyrogen free distilledwater or saline adjusted to an alkaline pH.

[0080] In order to enhance the effectiveness of the compound accordingto this invention, it is possible to use such adjuvants as given beloweither singly or in combination in accordance with the purpose of eachapplication thereof while taking into consideration the form of theirpreparation and their field of application.

[0081] Namely, exemplary adjuvants may include anionic surfactants suchas alkyl sulfates, aryl sulfonates, succinates, polyethylene glycolalkyl aryl ether sulfates, and the like, cationic surfactants such asalkylamines, polyoxyethylene alkylamines, etc., non-ionic surfactantssuch as polyoxyethylene glycol ethers, polyoxyethylene glycol esters,polyol esters and the like, and amphoteric surfactants. Encapsulation ormicroencapsulation of the active ingredient in liposome vesicles is alsowithin the scope of this invention.

[0082] Examples of stabilizers, thickeners, lubricants and the like areisopropyl hydrogen-phosphate, calcium stearate, wax, casein, sodiumalginate, serum albumin, other blood proteins, methylcellulose,carboxymethylcellulose, gum arabic, etc. It should be kept in mind thatthese ingredients are not limited to the recited examples.

[0083] The active materials according to the present invention can beadministered by any route, for example, orally, parenterally,intravenously, intradermally, subcutaneously, or topically, in liquid orsolid form. Preferably, the route of administration is intravenously.

[0084] The solutions or suspensions may also include the followingcomponents: a sterile diluent such as water for injection; salinesolution, oils, polyethylene glycols, glycerin, propylene glycol orother synthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple base vialsmade of glass or plastic.

[0085] While dosage values will vary with the specific severity of thedisease condition to be alleviated, good results are achieved when thecompounds described herein are administered to a subject requiring suchtreatment as an effective oral, parenteral or intravenous dose of from100 to 10,000 units per nanogram of measured endotoxin per day perpatient. A particularly preferred effective amount is about 1000 to 5000units per nanogram of measured endotoxin per day per patient. Mostpreferred is the administration of 0.1 to 100 mg of endotoxin bindingprotein/kg of body weight per day per patient. It is to be understood,however, that for any particular subject, specific dosage regimensshould be adjusted to the individual need and the professional judgmentof the person administering or supervising the administration of theaforesaid compound. It is to be further understood that the dosages setforth herein are exemplary only and they do not limit the scope orpractice of the invention. The dosages may be administered at once, ormay be divided into a number of smaller doses to be administered atvarying intervals of time.

[0086] Specific diseases which could be treated include septicemia,toxic shock, and any other condition, especially gram-negative bacterialinfections, which is accompanied by an increase in in vivo endotoxincontent. Other examples are endotoxin-related arthritis, gonorrhea,periodontal disease, spinal meningitis, and infections of amnioticfluid.

[0087] Although this invention and its preferred embodiments areprimarily addressed to use in humans, veterinary use is also encompassedby the invention. In this regard, the active ingredient may beadministered to reduce or prevent the pyrogenic or other ill effects ofendotoxin in vivo in, for example, dogs, cats, horses, cattle, sheep,and rabbits. Administration to laboratory animals, such as mice andrats, in order to prevent or reduce effects of endotoxin, is alsocontemplated.

[0088] Although polyclonal and monoclonal antibodies to endotoxin havealso been developed for this purpose and are the subject of ongoingdevelopment, the endotoxin binding protein of the present invention hasadvantages over antibodies which make it a preferred candidate for atherapeutic. The binding constant is very high and its low molecularweight may be less antigenic. In a recent paper, Greisman and Johnston,J. Infect. Disease 157:54-64 (1988), it has been shown that at least oneanti-serum against endotoxin is ineffective in reducing the effects ofendotoxin in vivo. In contrast, the in vivo experiments in the presentinvention have shown that the endotoxin binding protein may inactivateendotoxin in both rabbit and human serum.

[0089] Removal of Endotoxin From Solutions

[0090] Endotoxin contamination of pharmaceuticals is a problem towardwhich many removal protocols have been directed. Many of these aresummarized in Biopharm, April, 1988, pages 22-29, and the referencescited therein. They all fall short when the contaminated material is orcontains a high molecular weight substance, such as protein. Most of theexisting technology for endotoxin removal (ultrafiltration, ionexchange, affinity chromatography) does not separate protein andendotoxin. The present invention involves using the endotoxin bindingprotein (e.g., from Limulus) as an immobilized affinity ligand toachieve this separation. The preferred solid support for covalentlycoupling the endotoxin binding protein is cellulose, agarose or otherhydrophilic polymer derivatized to contain carboxyl, hydroxyl, amino,epoxide or other chemically reactive group to which proteins may becovalently attached using well known coupling chemistry. These methodsinclude using water soluble carbodiimide or carbonyldiimidazole,glutaraldehyde, etc. The most preferred support is the hydrophilic vinylpolymer in pellicular or membrane form containing carboxy-methyl groupsactivated with water-soluble carbodiimide. The inventors have attachedthe binding protein covalently to chromatographic beads and the proteinretains its biological activity. Furthermore, by mixing the beads withendotoxin contaminated proteins, the solution could be detoxified. Thiswas done by mixing the beads in batch mode in test tubes, or by packinga column with the beads and passing the solutions over it.

[0091] The protein could also be covalently attached to filter membranesfor the same purpose. FIG. 5 shows an experiment with such a filter. A40 ml solution containing approximately 130 pg/ml endotoxin wasrecirculated through a filter containing the binding protein. There wasan initial large drop in the toxin concentration that was furtherreduced as the material recirculated.

[0092] The present inventors further conducted a direct comparison oftwo immobilized polymyxin resins compared to the present ligand onsimilar gel particles. The experiment took 100 microliter gel volumes ofeach in a microcentrifuge tube and added 100 nanograms of E. coliendotoxin in 1 ml of various solutions to each. The tubes were mixed endover end for one hour. In 10 mM Tris, all three performed equally well.In 25% human serum albumin (HSA) and 5% HSA, the present endotoxinbinding protein was able to remove significantly more endotoxin.Interestingly, in distilled water, polymyxin was superior. Thus, thepresent endotoxin binding protein exhibits surprisingly superiorperformance in protein-containing solutions. Other possible proteinscontaminated by endotoxin are HSA, interferon, interleukins, growthfactors, hormones, proteases, TPA, TNF, monoclonals, EGF, insulin, anderythropoietin.

[0093] Extracorporeal Treatment of Septic Shock

[0094] Because of the ability of the endotoxin protein to function inprotein solutions and human serum, an affinity membrane or hollow fibermay be constructed for the purpose of removing endotoxin from the bloodof patients extracorporeally. In such a mode, similar to kidneydialysis, blood containing elevated concentrations of endotoxin from avariety of potential clinical conditions is circulated through anaffinity device such that the serum is brought into direct contact withendotoxin binding protein covalently bound to the membrane. Since theamount of endotoxin binding protein being released into the generalcirculation is much reduced from an intravenous application, potentialside effects are minimized.

[0095] Assay for Binding Affinity and Application of the EndotoxinBinding/Neutralizing Protein in an Alternative Endotoxin Assay

[0096] A known optical property of all proteins is the ability of somearomatic amino acids to fluoresce when excited at certain wavelengths.The present inventors looked specifically at tryptophan residues. Theseare excited at around 283 nm and fluoresce at around 350 nm. See FIG. 6.

[0097] The inventors examined a fluorescence maxima at 350 nm of theendotoxin binding protein at various concentrations of endotoxin. Therewas a proportional quenching of that fluorescence, indicative of a tightassociation. See FIG. 7. Other proteins as negative controls showed nosuch proportional change. See FIGS. 8 and 9. From this data, theinventors have been able to calculate the binding affinity constantsbetween the present protein and endotoxin. See FIGS. 9-12.

[0098] The fluorescence quenching experiment may serve as the basis foran assay for endotoxin based on the physical change of a single proteinupon exposure to endotoxin. The inventors have found that the quenchingeffect observed upon addition of endotoxin to the endotoxin bindingprotein does not appear to experience saturation up to a highconcentration level of added endotoxin. In other words, although onewould expect the relationship between concentration of endotoxin andquenching to be a nonlinear one, the linearity of the relationshipextends over a remarkably wide range of concentration of endotoxin. Thisunexpected aspect of the present assay enables the assay to be used overa wider range of endotoxin concentrations than could have been expected.Ranges of the ratio of endotoxin binding protein to endotoxin that areexpected to be effective are 0.1:1 to 5000:1, preferably 1:1 to 2000:1,most preferably 10:1 to 1000:1.

[0099] In a-preferred embodiment of the present endotoxin assay usingthe present endotoxin binding protein, the response is amplified using abiosensor. Biosensors rely on a specific binding ligand immobilized on asolid (e.g., quartz) chip. Electrical potential between two electrodes,or the acoustic wave perturbation between two electrodes is measured.

[0100] The current method for measuring endotoxin is the Limulusamebocyte lysate assay. This involves a cascade of enzymes which areactivated by endotoxin and result in the formation of a gel, analogousto a mammalian blood clot. Since multiple enzymes are involved, manysubstances interfere with the overall reaction, causing enhancement orinhibition. In addition, the key enzymes of the cascade are proteases soother proteolytic enzymes, such as trypsin, can cause gel formation. Toeliminate these problems, samples containing interfering substances mustbe diluted beyond the point where they interfere. In many cases thedilution factor can be hundreds or thousands. The net effect is todecrease the sensitivity in measuring absolute endotoxin concentrationsin those samples.

[0101] One of the promising potentials that a single endotoxin bindingprotein has is to eliminate the multiple interfering effects seen in theLAL assay. Even if the sensitivity were one tenth that of LAL, the lackof a need to dilute would compensate, making the sensitivity equivalent.Biosensors take advantage of specific binding affinities of suchmolecules. Antibodies are an example which have been used sound to thesurface of a silicon chip, they bind to their immunogen and cause achange in the surface which is measured electrically. Depending on thetype of sensor, capacitance, resistance or acoustic wave changes aremeasurable. Depending on the concentrations and volumes of the samples,the time required for each measurement can be from seconds to minutes.

[0102] The electronic component of the biosensor could measure voltage(potentiometric), current (amperometric), light, sound, temperature ormass (piezoelectric). The biosensors of the present invention can bebased on technology which is known, such as described in Biotechnology5, 437-440 (1987), and Transducers 1987, the abstract entitled“Development of a Surface Acoustic Wave Biosensor”, and “Recent Progressin Biosensor Development”, Hall, E. A. H., Int. J. Biochem. 1988, 20(4),357-62, each of which is incorporated herein by reference. Biosensorinformation is also contained in U.S. Pat. Nos. 4,721,677, 4,680,268, H0,000,201, and 4,614,714, each of which is incorporated herein byreference.

[0103] An actual device employing a biosensor would be similar to a flowcell. The sample would enter, be exposed to the reactive surface and ameasurement made. Depending on the chemistry of binding, the boundligand could be washed off to regenerate the surface or left on tomeasure a cumulative response. This may be adapted to an in-line deviceto monitor endotoxin contaminating events.

[0104] Due to the compact nature of the electronics, a portable fieldunit is feasible. This could be used on-site for checking water purityor process cleanliness during pharmaceutical manufacture, kidneydialysis unit pyrogen checks, or any other application where theconventional LAL is used now. Since endotoxin is a component of gramnegative bacterial cell walls, binding of whole bacteria should bemeasurable. An extension of this technology should make possible remotesensors to monitor water quality in the environment.

[0105] By coupling the binding protein to latex microspheres using achemistry similar to that used to immobilize the protein onchromatographic resins or membranes, another method to quantitateendotoxin is constituted. By exposing latex microspheres coated withendotoxin binding protein (e.g., from Limulus) to endotoxin, themicrospheres agglutinate. Such agglutination is commonly employed inassays based on antibodies to the specific ligand to be measured(Hechemy, K. E.; Michaelson, E. E. Laboratory Menacement 1984,22(6):27,ff (Part I) and 22(7):26,f (Part II); Babson, A. L., Opper, C.A. and Crane L. J. American Journal of Clinical Pathology (1982),77(4):424-9). In such techniques, the agglutination phenomenon isthought to be due to crosslinking between multiple binding sites on theantibody molecule complexing with multiple sites on the antigen. Due tothe high ratio of endotoxin binding protein to endotoxin needed to beeffective in our other solution experiments, it is thought the number ofendotoxin binding sites on the protein is one or less. By covalentlycoupling many molecules of binding protein per microsphere, a multiplebinding site reagent is created which is now possible to function as aspecific agglutinin. Alternatively, the binding protein can be linked toone suspension of beads and endotoxin or lipid A linked to a secondsuspension. On mixing, these two suspensions will agglutinate. This mayalso be employed as an assay for endotoxin in solution by the ability ofthe free endotoxin to inhibit such agglutination. In this case,agglutination is inversely proportional to the unknown endotoxinconcentration.

[0106] The invention now being generally described, it will now beillustrated in greater detail by the following examples, which arepresented herein for illustration only and are not intended to belimiting of the present invention, unless so indicated.

EXAMPLES Example 1

[0107] Assay in of in vitro Inactivated Endotoxin by Pyrogen Testing inRabbits:

[0108]E. coli endotoxin was prepared at 100 nanograms per ml. InPhosphate Buffered Saline (PBS). This was mixed with 200 micrograms ofLimulus Endotoxin Binding Protein and incubated at 37 degrees Celsiusfor one hour. Control solutions were prepared of endotoxin (100nanograms/ml) only and PBS only. Three vials of each of the threesolutions were prepared containing 2 ml/vial. 1.5 ml of each solutionwas injected intravenously into individual 3 kilogram rabbits and theirbody temperatures measured continuously for six hours with data pointsrecorded every 10 minutes. The final dose of endotoxin or inactivatedendotoxin was 50 nanograms/kilogram.

[0109] In rabbits, a 5 nanograms/kilogram dose of endotoxin elicits ameasurable fever response, showing a peak at one hour after injection.The present results showed the rabbits receiving only endotoxin at 50nanograms/kilogram did indeed develop a body temperature elevated 1.64(S.D. 0.236) degrees Celsius. Those rabbits receiving only PBS orendotoxin inactivated with Limulus Endotoxin Binding Protein maintaineda normal body temperature.

Example 2

[0110] Assay of in vivo Prophylactic Efficacy of Limulus EndotoxinBinding Protein:

[0111] Limulus Endotoxin Binding Protein is injected intravenously intorabbits at two dose levels, 5 micrograms/kilogram and 50micrograms/kilogram. Control animals receive injections of PBS. Fifteenminutes later, all animals receive intravenous injections of 50nanograms/kilogram E. coli endotoxin. Body temperature is monitoredcontinuously over six hours and recorded every 10 minutes. The normalcourse of increase in temperature peaking at one hour after injection isseen in control animals preinjected with PBS only. Those animalsreceiving preinjection of endotoxin binding protein at 50micrograms/kilogram showed an increase of 1.55 (S.D. 0.225) degreesCelsius. Animals receiving 5 micrograms/kilogram demonstratedtemperature increases of 1.85 (S.D. 0.270).

Example 3

[0112] Assay of in vivo Therapeutic Efficacy of Limulus EndotoxinBinding Protein:

[0113]E. coli endotoxin is injected intravenously into 9 rabbits at adose of 50 nanograms/kg. After 15 minutes, three were injectedintravenously with 5 micrograms Limulus Endotoxin Binding Protein, threewere injected intravenously with 50 micrograms Limulus Endotoxin BindingProtein and three received PBS. Volumes of all injections were 0.5ml/kg. Body temperatures are monitored for 6 hours and data collectedevery 10 minutes. Animals receiving endotoxin and the PBS only, manifestthe normal peak fever response one hour after toxin administration.Those animals receiving therapeutic injections of the Limulus proteinexhibit a much reduced fever response proportional to the amount ofprotein which is administered.

Example 4

[0114] Endotoxin Inactivating Potential of Limulus Endotoxin BindingProtein in Human Serum:

[0115] In order to assay the potential effectiveness of the endotoxinbinding protein as a therapeutic or prophylactic agent for septic shockor related disorders in humans, the protein was tested for its abilityto inactivate endotoxin in the presence of whole human serum. The assaywas conducted in a 96-well tissue culture multiwell elates as describedin detail in Novitsky et al., J. Clin. Micro., 20: 211-216 (1985). Toeach well were added-in order 0.05 ml of serum only or serum with 25micrograms/ml Limulus Endotoxin Binding Protein, 0.05 ml E. coliendotoxin solution. The plates were covered with Parafilm to preventevaporation, agitated on a mechanical vibrating platform for 15 secondsand incubated at 37 degrees Celsius. Multiwell plates were thenuncovered, and 0.1 ml of LAL was added to each well. The plates weresubsequently handled and read as described for the LAL endotoxin assay.Serum with the added endotoxin binding protein was able to inactivateincreasingly larger amounts of endotoxin (2,000 nanograms/ml) whileserum alone was able to bind and/or inactivate only 80 nanograms/ml.

Example 5

[0116] Universality of Endotoxin Type Inactivated by the LimulusEndotoxin Binding Protein.

[0117] In order to investigate the mechanism of the endotoxin bindingphenomenon and determine the breadth of endotoxin types against whichthe binding protein is effective, endotoxin from several species of Gramnegative bacteria as well as lipid A were tested. These includedendotoxin from Klebsielia pneumonias, Serratia marcescens, Salmonellaenteritidis, Escherichia coli 0113 wild type, E. coli rough mutant(J-5), Salmonella abortus eaui, and Lipid A from S. minnesota Re 595.

[0118] The endotoxin binding protein was mixed with the endotoxins orlipid A at a ratio of 5:1 to 1,000:1 in the presence of 10 millimolarTris buffer, pH 6-8. In all cases the measurable endotoxin activityafter mixing was reduced 85% to 99.5%.

Example 6

[0119] Summary:

[0120] Limulus Endotoxin Binding Protein (EBP) protects rats from thelethal effects of lipopolysaccharide (LPS). The effects of premixed LPSand EBP(1:1 wt/wt) were compared to those of EBP or LPS alone. Thematerial was administered to rats in groups. The endotoxin groupexhibited 30% mortality; the group which received the combined EBP+LPSexhibited no deaths. In an in vitro experiment, vascular tissue wasexamined for contraction defects after preincubation with endotoxin,protein, or a mixture of endotoxin and protein. Endotoxin-incubatedtissue exhibited reduced contraction to norepinephrine; coincubationwith limulus protein protected against the supression.

[0121] Hematocytes from Limulus polyphemus contain a 12,000 daltonamphipathic protein with a high affinity for the lipid A portion ofendotoxin. This protein is part of an anti-infection pathway ofaggregation where rapid degranulation and clot formation are initiatedwhen hematocytes are exposed to Gram-negative endotoxins. This endotoxinbinding protein has been isolated and sequenced (SEQ. ID. NO. I). Invitro it binds to endotoxin with high affinity and neutralizes thelipopolysaccharide thus preventing it from being detected in the Limulusamebocyte assay. In a recent study which shows that this protein canprotect endothelial cells in vitro from the toxic affect of endotoxin,the inventors examined the ability of the Limulus endotoxinbinding-protein to protect rats from the toxic effects of endotoxin.Also, the ability of this protein to protect vascular tissue from theeffects of endotoxin was examined in vitro experiments. The results ofboth in vivo and in-vitro experiments demonstrate that this proteinexhibits a protective effect by neutralizing the endotoxin.

[0122] Methods:

[0123] Protection From Endotoxemia

[0124] Male Sprague-Dawley rats (230-450 g) were given light Halothaneanesthesia and then injected with the appropriate experimental treatmentvia the dorsal vein of the penis. Groups of rats received one of thefollowing treatments;

[0125] 1) LPS (E. coli 0111:B4, Sigma Chemical Company) in buffer, 0.15M NaCl buffered to pH 7.4 with 0.02 M sodium phosphate,

[0126] 2) A suspension of anti-LPS factor and LPS mixed 1:1 (wt/wt) inbuffer,

[0127] 3) Albumin mixed with LPS 1:1 (wt/wt), or

[0128] 4) only the Limulus protein incubated in buffer.

[0129] All solutions were incubated at 37° C. for one hour prior toinjection. The volume injected (1.7 to 2.9 ml) depended on the weight ofthe rat and was adjusted to deliver an exact dose on a mg LPS/kg bodyweight basis. Animals were maintained on standard rat chow and water adlibitum. Survival was followed to 24 hours.

[0130] Aortic Ring Contraction

[0131] Rats were sacrificed by decapitation and the thoracic aortaexcised and sectioned into rings for measures of contractileperformance. Tissues from normal rats were incubated at 37° C. for 16hours in DMEM (Dulbecco's Minimal Essential Medium) containing 5% fetalcalf serum, 100 U penicillin, 100 ug streptomycin, and gassed with 95%CO₂-5% CO₂. Experiments consisted of additions of either;

[0132] 1) 10 ng/ml endotoxin,

[0133] 2) 50 ng/ml endotoxin binding-protein,

[0134] 3) a mixture of endotoxin and binding-protein (IL0 and 50 ng/ml),or

[0135] 4) medium only during the 16 hr incubation period.

[0136] Contractile performance of rings suspended between two hooks in a10 ml bath was assessed by measuring the tension as a function ofcumulative doses of norepinephrine (NE) covering a range of 1 nM to 30uM. Survival as a Function of Endotoxin/Neutralizing Protein (EBP)Treatment # Survivors/# Rats % Survival EXP¹ # 1 LPS 14/20  70  LPS +albumin  9/20  40  {close oversize bracket} Δ 30 LPS + EBP 20/20100^(a,b) EBP (only) 13/15  87  LPS = (15 mg/kg, # 37F-4089) this lot #exhibits reduced toxicity ^(a)p < .05 different from LPS Buffer ^(b)p <.05 different from LPS albumin EXP² # 2 LPS  9/28  32 LPS + albumin 7/28  25 {close oversize bracket} Δ 18 LPS + EBP 14/27 EBP (only) 11/11100 EBP (only) 11/11 100 ²LPS = (15 mg/kg, # 39F-4030 and # 37F-4019)all proteins = 15 mg/kg ^(c,d) no significant different but trendconsistent (p < .1) Survival as a Function of NativeEndotoxin/Neutralizing Protein (nENP) and Recombinant EndotoxinBinding/Neutralizing Protein (rENP) Treatment # Survivors/# Rats %Survival LPS  3/10  30 LPS + albumin  5/10  50 {close oversize bracket}Δ 70 LPS + r-EBP 10/10 100^(d,c) EBP (only) 10/10 100 LPS = (15 mg/kg, #39F-4030) all proteins = 15 mg/kg ^(d)p < .05 different from LPS inbuffer ^(e)p < .05 different from LPS in albumin * * * * *

[0137] The invention now being fully described, it will be appreciatedthat the invention may be practiced otherwise than as specifically setforth herein.

What is new and desired to be secured by Letters Patent of the UnitedStates is:
 1. A method for isolating an endotoxin binding protein from ahorseshoe crab, which comprises: subjecting amebocytes obtained from ahorseshoe crab to hypotonic shock to lyse said amebocytes, and obtaincell debris from said lysed amebocytes, extracting said cell debris witha solution containing a denaturant selected from the group consisting ofurea and guanidine hydrochloride, to produce an extract, passing saidextract through a first ultrafiltration membrane having a molecularcutoff of from 20,000 to 50,000 daltons, to obtain a filtrate,concentrating said filtrate by passing it through a secondultrafiltration membrane having a molecular cutoff of from 5,000 to10,000, to produce a retentate, subjecting said-retentate to cationexchange chromatography at a pH of from about 5 to 6, using an elutionbuffer which comprises urea, and eluting a solution containing a peak ofendotoxin binding activity, applying said solution containing said peakof endotoxin binding activity to a reverse phase column, and adding abuffer to said reverse phase column, to obtain a solution containingpurified endotoxin binding protein.
 2. The method according to claim 1,wherein said hypotonic shock is accomplished by treating said amebocyteswith endotoxin-free distilled water at about 0° C. to 10° C.
 3. Themethod according to claim 1, wherein said extraction of said cell debrisis accomplished with 6 molar urea or guanidine hydrochloride.
 4. Themethod according to claim 1, wherein said ultrafiltration membranes areeach composed of polysulfone.
 5. The method according to claim 1,wherein before passing said extract through said membrane, the extractis crudely filtered with a filter aid selected from the group consistingof diatomaceous earth, cationic and anionic colloidal particlesuspensions.
 6. The method according to claim 4, wherein the firstpolysulfone membrane has a molecular cutoff of 30,000 daltons.
 7. Themethod according to claim 4, wherein the second polysulfone membrane hasa molecular cutoff of 8,000 daltons.
 8. The method according to claim 1,wherein said cation exchange chromatographic step is accomplished withSepharose.
 9. The method according to claim 1, wherein said cationexchange step involves elution from said cation exchange column with astep gradient of a salt selected from the group consisting of ammoniumchloride, potassium chloride, and sodium chloride.
 10. The methodaccording to claim 1, wherein said cation exchange chromatographic stepincludes elution with a buffer containing 1 to 6 molar urea.
 11. Themethod according to claim 1, wherein said reversed phase column is aresin having 4, 8, or 18 carbon atom chains.
 12. The method according toclaim 1, wherein said reverse phase column is eluted with a stepgradient of isopropanol and trifluoroacetic acid.
 13. The methodaccording to claim 12, wherein said trifluoroacetic acid has aconcentration ranging from 0.15 to 0.25%.
 14. The method according toclaim 1, wherein said horseshoe crab is Limulus polyphemus.
 15. Aproduct produced by the method of claim 1, wherein said product hasendotoxin binding capability, and is a protein having an initial aminoacid sequence selected from the group consisting ofSer-Asn-Ile-Trp-Thr-, Asp-Asn-, Ser-Gly-, and Ser-Asn-.
 16. A method forameliorating the biological effects of endotoxin in vivo, whichcomprises administering to a mammal in need of such treatment aneffective amount of an endotoxin-binding protein of the horseshoe crab.17. The method of claim 16, wherein said endotoxin-binding protein hasan initial amino acid sequence selected from the group consisting ofSer-Asn-Ile-Trp-Thr-, Asp-Asn-, Ser-Gly-, and Ser-Asn.
 18. A method forameliorating the biological effects of endotoxin in vivo, whichcomprises administering to a mammal in need of such treatment aneffective amount of an endotoxin-binding protein preparation obtainedfrom the horseshoe crab according to the following process: (a)subjecting amebocytes obtained from a horse-shoe crab to hypotonic shockto lyse said amebocytes, and obtain cell debris from said lysedamebocytes; (b) extracting said cell debris with a solution containing adenaturant selected from the group consisting of urea and guanidinehydrochloride, to produce an extract; (c) passing said extract through afirst ultra-filtration membrane having a molecular cutoff of from 20,000to 50,000 daltons, to obtain a filtrate; (d) concentrating said filtrateby passing it through a second ultrafiltration membrane having amolecular cutoff of from 5,000 to 10,000, to produce a retentate; (e)subjecting said retentate to cation exchange chromatography at a pH offrom about 5 to 6, using an elution buffer which comprises urea, andeluting a solution containing a peak of endotoxin binding activity; and(f) applying said solution containing said peak of endotoxin bindingactivity to a reverse phase column, and adding a buffer to said reversephase column, to obtain a solution containing purified endotoxin bindingprotein.
 19. The method according to claim 18, wherein said hypotonicshock is accomplished by treating said amebocytes with endotoxin-freedistilled water at about 0° C. to 10° C.
 20. The method according toclaim 18, wherein said extraction of said cell debris is accomplishedwith 6 molar urea or guanidine hydrochloride.
 21. The method accordingto claim 18, wherein said ultrafiltration membranes are each composed ofpolysulfone.
 22. The method according to claim 18, wherein beforepassing said extract through said membrane, the extract is crudelyfiltered with a filter aid selected from the group consisting ofdiatomaceous earth, cationic and anionic colloidal particle suspensions.23. The method according to claim 21, wherein the first polysulfonemembrane has a molecular cutoff of 30,000 daltons.
 24. The methodaccording to claim 21, wherein the second polysulfone membrane has amolecular cutoff of 8,000 daltons.
 25. The method according to claim 18,wherein said cation exchange chromatographic step is accomplished withSepharose.
 26. The method according to claim 18, wherein said cationexchange step involves elution from said cation exchange column with astep gradient of salt selected from the group consisting of ammoniumchloride, potassium chloride, and sodium chloride.
 27. The methodaccording to claim 18, wherein said cation exchange chromatographic stepincludes elution with a buffer containing 1 to 6 molar urea.
 28. Themethod according to claim 18, wherein said reverse phase column is aresin having 4, 8, or 18 carbon atom chains.
 29. The method according toclaim 18, wherein said reverse phase column is eluted with a stepgradient of isopropanol and trifluoroacetic acid.
 30. The methodaccording to claim 29, wherein said trifluoroacetic acid has aconcentration ranging from 0.15 to 0.25%.
 31. The method according toclaim 18, wherein said horseshoe crab is Limulus polyphemus.
 32. Themethod according to claim 16 or 18, wherein said endotoxin bindingprotein is administered intravenously.
 33. The method according to claim16 or 18, wherein said mammal is a human.
 34. The method according toclaim 16 or 18, wherein the dose of endotoxin binding protein is fromabout 0.1 to 100 mg of endotoxin binding protein per kg of body weightper day per patient.
 35. A pharmaceutical composition effective foramelio-rating the biological effects of endotoxin in vivo whenadministered to a mammal, comprising a purified endotoxin-bindingprotein of the horseshoe crab and a pharmaceutically acceptable carrier.36. The pharmaceutical composition of claim 35, where-in saidendotoxin-binding protein has an initial amino acid sequence selectedfrom the group consisting of Ser-Asn-Ile-Trp-Thr- Asp-Asn-, Ser-Gly-,and Ser-Asn.
 37. A pharmaceutical composition effective foramelio-rating the biological effects of endotoxin in vivo whenadministered to a mammal, comprising a purified endotoxin-bindingprotein preparation and a pharmaceutically acceptable carrier, whereinsaid protein preparation is obtained according to the following process:(a) subjecting amebocytes obtained from a horse-shoe crab to hypotonicshock to lyse said amebocytes, and obtain cell debris from said lysedamebocytes; (b) extracting said cell debris with a solution containing adenaturant selected from the group consisting of urea and guanidinehydrochloride, to produce an extract; (c) passing said extract through afirst ultra-filtration membrane having a molecular cutoff of from 20,000to 50,000 daltons, to obtain a filtrate; (d) concentrating said filtrateby passing it through a second ultrafiltration membrane having amolecular cutoff of from 5,000 to 10,000, to produce a retentate; (e)subjecting said retentate to cation exchange chromatography at a pH offrom about 5 to 6, using an elution buffer which comprises urea, andeluting a solution containing a peak of endotoxin binding activity; and(f) applying said solution containing said peak of endotoxin bindingactivity to a reverse phase column, and adding a buffer to said reversephase column, to obtain a solution containing purified endotoxin bindingprotein.
 38. The pharmaceutical composition according to claim 37,wherein said hypotonic shock is accomplished by treating said amebocyteswith endotoxin-free distilled water at about 0° C. to 10° C.
 39. Thepharmaceutical composition according to claim 37, wherein saidextraction of said cell debris is accomplished with 6 molar urea orguanidine hydrochloride.
 40. The pharmaceutical composition according toclaim 37, wherein said ultrafiltration membranes are each composed ofpolysulfone.
 41. The pharmaceutical composition according to claim 37,wherein before passing said extract through said membrane, the extractis crudely filtered with a filter aid selected from the group consistingof diatomaceous earth, cationic and anionic colloidal particlesuspensions.
 42. The pharmaceutical composition according to claim 40,wherein the first polysulfone membrane has a molecular cutoff of 30,000daltons.
 43. The pharmaceutical composition according to claim 40,wherein the second polysulfone membrane has a molecular cutoff of 8,000daltons.
 44. The pharmaceutical composition according to claim 37,wherein said cation exchange chromatographic step is accomplished withSepharose.
 45. The pharmaceutical composition according to claim 37,wherein said cation exchange step involves elution from said cationexchange column with a step gradient of salt selected from the groupconsisting of ammonium chloride, potassium chloride, and sodiumchloride.
 46. The pharmaceutical composition according to claim 37,wherein said cation exchange chromatographic step includes elution witha buffer containing 1 to 6 molar urea.
 47. The pharmaceuticalcomposition according to claim 37, wherein said reverse phase column isa resin having 4, 8, or 18 carbon atom chains.
 48. The pharmaceuticalcomposition according to claim 37, wherein said reverse phase column iseluted with a step gradient of isopropanol and trifluoroacetic acid. 49.The pharmaceutical composition according to claim 48, wherein saidtrifluoroacetic acid has a concentration ranging from 0.15 to 0.25%. 50.The pharmaceutical composition according to claim 37, wherein saidhorseshoe crab is Limulus polyphemus.
 51. A method for ameliorating thebiological effects of endotoxin in vivo, which comprises administeringto a mammal in need thereof an effective amount of an endotoxin bindingprotein having amino acid sequence SEQ. ID. NO. I.
 52. The methodaccording to claim 51, wherein said endotoxin binding protein isadministered intravenously.
 53. The method according to claim 51,wherein said mammal is a human.
 54. The method according to claim 53,wherein the dose of endotoxin binding protein is from about 0.1 to 100mg of endotoxin binding protein per kg of body weight per day perpatient.
 55. The method according to claim 51, wherein said endotoxinbinding protein is administered prophylactically to said mammal beforethe mammal is exposed to endotoxin.
 56. The method according to claim51, wherein said endotoxin binding protein is administered to saidmammal after the mammal is exposed to endotoxin.
 57. A pharmaceuticalcomposition for administration to a mammal to ameliorate the biologicaleffects of endotoxin in vivo, which comprises an endotoxin bindingprotein having amino acid sequence SEQ. ID. NO. I in combination with apharmaceutically acceptable carrier material.
 58. A method forameliorating the biological effects of endotoxin in vivo which comprisesadministering to a mammal in need thereof an effective amount of anendotoxin binding protein having an amino acid sequence corresponding atleast to amino acid position 30 to 55 of SEQ. ID. NO. III and up tocomplete SEQ. ID. NO. III.
 59. The method according to claim 58, whereinsaid endotoxin binding protein is administered intravenously.
 60. Themethod according to claim 58, wherein said mammal is a human.
 61. Themethod according to claim 59, wherein the dose of endotoxin bindingprotein is from about 0.1 to 100 mg of endotoxin binding protein per kgof body weight day per patient.
 62. The method according to claim 58,wherein said endotoxin binding protein is administered prophylacticallyto said mammal before the mammal is exposed to endotoxin.
 63. The methodaccording to claim 58, wherein said endotoxin binding protein isadministered to said mammal after the mammal is exposed to endotoxin.64. A pharmaceutical composition for administration to a mammal toameliorate the biological effects of endotoxin in vivo, which comprisesan endotoxin binding protein having an amino acid sequence correspondingat least to amino acid positions 30 to 55 of SEQ. ID. NO. III and up tocomplete amino SEQ. ID. NO. III, in combination with a pharmaceuticallyacceptable carrier material.
 65. An endotoxin binding protein havingamino acid sequence SEQ. ID. No. I free of the contaminating componentsnaturally associated with the horseshoe crab.
 66. An endotoxin bindingprotein having an amino acid sequence corresponding at least to aminoacid positions 30 to 55 of SEQ. ID. No. III and up to complete aminoSEQ. ID. NO. III free of the contaminating components naturallyassociated with the horseshoe crab.
 67. A DNA molecule coding for anendotoxin binding protein having amino acid sequence SEQ. ID. NO. I,wherein said DNA molecule is free of the contaminating componentsnaturally associated with the horseshoe crab.
 68. A DNA molecule codingfor an endotoxin binding protein having an amino acid sequencecorresponding at least to amino acid positions 30 to 55 of SEQ. ID. NO.III and up to complete amino SEQ. ID. NO. III, wherein said DNA moleculeis free of the contaminating components naturally associated with thehorseshoe crab.
 69. The endotoxin binding protein of claim 15, 65 or 66immobilized on a solid phase support.
 70. The immobilized endotoxinbinding protein of claim 69, wherein said solid phase support is achromato-graphic resin or a membrane.
 71. The immobilized endotoxinbinding protein of claim 69 which is a biosensor device.
 72. Thebiosensor device of claim 71, wherein said solid phase support is quartzor silicon.
 73. A method for assaying endotoxin concentration, whichcomprises contacting serial aqueous dilutions of a material suspected ofcontaining endotoxin with a known quantity of the endotoxin bindingprotein of claim 15, 65 or 66, observing the fluorescence emission of atleast one wavelength of the endotoxin binding protein before and aftercontact with the aqueous dilutions of said material suspected ofcontaining endotoxin, correlating the levels of fluorescence emissionwith at least one known emission level to thereby determine the quantityof endotoxin present in said material suspected of containing endotoxin.74. The method of claim 73, wherein said material is a body fluid. 75.The method of claim 73, wherein said fluorescence emission of theendotoxin binding protein is measured at from 340 to 360 nanometers. 76.The method of claim 73, wherein said fluorescence emission is producedby excitation at from about 275 to 295 nanometers.
 77. A method forreducing endotoxin contamination of a material suspected of containingendotoxin, comprising contacting said material with the endotoxinbinding molecule of claim 15, 65 or 66 to form a complex betweenendotoxin and the endotoxin binding molecule, and separating saidcomplex from said sample.
 78. The method of claim 77, wherein saidendotoxin binding protein is immobilized on a chromatographic resin oron a membrane.
 79. A method for the extracoporeal removal of endotoxinfrom blood, comprising contacting blood with the immobilized endotoxinbinding protein of claim
 69. 80. A method for assaying for endotoxinconcentration in a material suspected of containing endotoxin, whichcomprises contacting said material with a biosensor device comprisingthe endotoxin binding protein of claim 15, 65 or 66 immobilized on asolid phase support, detecting a change in capacitance, resistance, oracoustic wave of said solid phase support, and correlating the changewith the changes observed with standard solutions of endotoxin bindingprotein.
 81. The method of claim 80, wherein said solid phase port isquartz or silicon.